This uncommon colitis is caused by Clostridium difficile toxin released into
the gut. It is most commonly associated with antibiotic therapy which destroys
or inhibits much of the normal bacterial flora of the gut, allowing overgrowth
of the Clostridium species with release of the toxin. It is this toxin that
is assayed in the most common laboratory tests.

Pharmacy Practice Research Group, School of Pharmacy, The Queen's
University of Belfast, Northern Ireland.

J Clin Pharm Ther 2000 Apr;25(2):101-9 Abstract quote

OBJECTIVE: The aim of the present study was to evaluate the incidence,
risk factors and cost implications of Clostridium difficile-associated
diarrhoea (CDAD) in hospitalized adult patients.

METHODS: Eighty-seven hospitalized adult patients, positively identified
as having CDAD, were reviewed retrospectively to determine the risk
factors and cost implications of CDAD.

RESULTS: The clinical manifestations, in addition to diarrhoea, included
elevated temperature (= 37.8 degrees C; 42.5%), abdominal pain (63.
2%) and leucocytosis (=12 x 109 cells/l; 52.9%). Eight patients underwent
endoscopy, and pseudomembranous colitis was confirmed in all of these
patients. Nine patients died during their hospital stay. Cefotaxime
and cefuroxime were the agents most commonly associated with CDAD. There
was a significant difference (P < 0.001) between the sex distribution
of CDAD patients and adult hospital patients (69% of CDAD patients were
female vs. 52% of general adult hospital population). Significantly
(P < 0.001) more patients with CDAD were admitted from the nursing
home (NH) setting. The mean age of patients with CDAD admitted from
NHs (n = 19) was older than those cases admitted from the community
(n = 68) by 14 years (P < 0.001). The length of hospital stay was
significantly (P < 0.001) longer for patients with CDAD (16.9 vs.
3.89 days). No differences (P = 0.306) were found in the response times
for CDAD patients treated with either oral metronidazole (n = 39) or
oral vancomycin (n = 48). The mean response time was, however, significantly
longer in the CDAD patients admitted from NHs (4.2 days) compared with
those admitted from the community (2.5 days), although the former patients
were older and had significantly more comorbidity (P < 0.001). The
mean cost per one treated-case of CDAD (bed, laboratory requests and
treatment therapy) was calculated as pound2860.

CONCLUSION: Patients admitted from NHs are at increased risk of development
of CDAD; receiving cefotaxime or cefuroxime axetil (oral form), being
elderly and being female are risk factors for the development of CDAD.
Treatment of CDAD with oral metronidazole or oral vancomycin gives rise
to similar response times and efficacy.

Department of Infectious Diseases, University Hospital of Umea,
Sweden.

J Antimicrob Chemother 2001 Jan;47(1):43-50 Abstract quote

The frequency of antibiotic-associated diarrhoea (AAD) and Clostridium
difficile-associated diarrhoea (CdAD) was prospectively determined in
a population of 2462 patients recruited from five Swedish hospitals,
including divisions for infectious diseases, orthopaedics, surgery,
geriatrics, nephrology and internal medicine. AAD developed in 4.9%
of the treated patients.

Faecal samples were obtained from 69% of patients with AAD and 55.4%
were positive for C. difficile cytotoxin B. The frequency of AAD varied
from 1.8 to 6.9% at the participating centres (P < 0.001). The frequency
of AAD also varied considerably between medical disciplines and wards
within different hospitals and was highest in the nephrology and geriatric
units (6.7 and 7.1%, respectively). There was no difference in frequency
of AAD when analysed with respect to gender or age. Medical interventions
(laxative treatment, endoscopy and abdominal surgery) or presence of
one concomitant disease (diabetes, malignancy, chronic renal disease
and inflammatory bowel disease) did not significantly affect the frequency
of AAD, whereas patients suffering from two or more of these illnesses
had significantly (P = 0.001) higher frequencies of AAD. Patients treated
with antibiotics for 3 days had a significantly (P = 0.009) lower frequency
of AAD than those treated for longer periods.

Treatment with cephalosporins, clindamycin or broad-spectrum penicillins
was associated with an increased risk of AAD. With specimens from one
centre, 62.5% of tested patients with AAD and 33.8% of asymptomatic
patients were positive for cytotoxin B. Although C. difficile cytotoxin
B in stool samples was significantly associated with AAD (P = 0.003),
the causal relationship with diarrhoea is not always evident.

Clostridium difficile cytotoxin B in adults with diarrhea: a comparison
of patients treated or not treated with antibiotics prior to infection.

OBJECTIVE: To study the detection rate of Clostridium difficile cytotoxin
B in stool specimens from adults with diarrhea as related to previous
antimicrobial treatment.

METHODS: Stool specimens from 802 adult patients with diarrhea and
203 healthy controls were tested for C. difficile cytotoxin B using
a cell cytotoxicity assay. Antibiotic susceptibility testing of C. difficile
was performed with the E test.

RESULTS: Of 173 patients treated with antimicrobial medication within
5 weeks of onset of diarrhea, 60 (35%) were positive for C. difficile
cytotoxin B (group A) compared to only 41 (7%) of 629 untreated patients
(group B) and two of the 203 (1%) healthy controls. Compared to patients
in group A, patients in group B possessed characteristics not usually
connected with C. difficile disease. They were generally younger (median
age 40 years vs. 73 years), had been hospitalized less frequently (10%
vs. 67%), had more often travelled abroad within the previous 2 weeks
(46% vs. 1%), and more often had multiple enteropathogens (41% vs. 3%).
Minimal inhibitory concentrations for vancomycin, metronidazole and
fucidic acid to C. difficile isolates ranged from 0.5 to 4 mg/L, from
0.125 to 256 mg/L and 0.25 to 4 mg/L, respectively.

CONCLUSIONS: The detection rate of C. difficile cytotoxin B in patients
with diarrhea, not associated with antibiotic treatment, is comparable
to that in healthy control subjects. It probably merely reflects a carrier
state without clinical significance.

GEOGRAPHY

FRANCE

Prevalence and Genetic Characterization of Toxin A Variant Strains of
Clostridium difficile among Adults and Children with Diarrhea in France.

Toxin A variant strains (toxin A-negative, toxin B-positive strains)
of Clostridium difficile have been reported to be responsible for diarrhea
or pseudomembranous colitis in humans. These strains lack parts of the
repeating sequences of the toxin A gene (tcdA) and are toxin A negative
by commercial enzyme immunoassays (EIA).

Here, we report the prevalence of the toxin A variant strains in 334
patients with C. difficile-associated diarrhea in France. The repeating
segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers
NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998).
In the case of amplified fragments of unexpected size, the entire tcdA
gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol.
36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field
gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and
NK11, C. difficile variant strains were detected in 2.7% of patients.
Several variant types were found. A deletion of approximately 1,700
bp was observed in six strains from five patients. These strains belonged
to serotype F and were characterized by the same pulsotype and the same
PCR ribotype. They were toxin A negative by EIA and exhibited an atypical
cytopathic effect on MRC-5 cells.

Two other tcdA variant types that exhibited a positive result for toxin
A by EIA were identified: one from serotype H with a longer amplified
fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis
of C. difficile-associated diseases would have been missed in five patients
(1.5%) by laboratories that screen the stools only for the presence
of toxin A.

This result underlines the need for testing stool by the cytotoxicity
assay in patients with a high suspicion of C. difficile-associated diarrhea
but a negative immunoassay for toxin A.

Department of Infectious Diseases, University of Lund, University
Hospital, Malmo, Sweden.

J Hosp Infect 1999 Dec;43(4):265-73 Abstract quote

An increased prevalence of patients with C. difficile-associated diarrhoea
in a hospital setting suggested the possible existence of an endemic
occurrence.

A study was therefore designed to determine clonal relatedness among
173 isolates of C. difficile, collected consecutively during 1995 from
147 patients (89 inpatients and 58 outpatients) and to estimate the
probability of nosocomial transmission. Arbitrarily primed PCR (AP-PCR)
with three different primers, AP1, AP2 and CLD1, was used for fingerprinting
and identified 21, 92 and 70 types, respectively. Overall DNA analysis
of the combined AP-PCR data yielded 140 types, of which 130 were unique,
whereas 10 types occurred repeatedly in 36 isolates from 33 patients;
seven isolates were non-typeable by one of the primers.

Epidemiological data confirmed that in eight of the 33 patients there
was a high probability of nosocomial transmission. Despite a high prevalence
of C. difficile among hospitalized patients, a low frequency of nosocomial
transmission was suggested by high resolution molecular typing of bacterial
isolates in conjunction with traditional epidemiological methods.

DISEASE ASSOCIATIONS

CHARACTERIZATION

ANTIBIOTIC TREATMENT

Factors associated with nosocomial diarrhea and Clostridium difficile-associated
disease on the adult wards of an urban tertiary care hospital.

A prospective survey of the adult inpatient population of an urban
tertiary care hospital was conducted to determine factors associated
with the development of nosocomial diarrhea and the acquisition of Clostridium
difficile-associated disease. During the 3-month survey, 98 patients
with nosocomial diarrhea were enrolled, and 38 controls were recruited.

The controls were patients without diarrhea lying in beds adjacent
to the affected patients. Factors significantly associated with nosocomial
diarrhea were the administration of a special diet (P = 0.02) and receipt
of a greater number of different antibiotics (P=0.02). Among the 98
patients with diarrhea, Clostridium difficile toxin B was identified
in the stool of 13. Factors found to be associated with the presence
of toxin B as compared to other causes of nosocomial diarrhea were a
greater number of individual antibiotics used during hospitalization
(P=0.02) and receipt of a cephalosporin (P=0.03) or, more specifically,
a third-generation cephalosporin (P=0.02).

Among patients with nosocomial diarrhea, those who had toxin in their
stool had a significantly higher total antibiotic burden (expressed
as antibiotic days) than those with diarrhea due to other causes (P
= 0.01).

Synchronous collagenous and pseudomembranous colitis has not been previously
reported. A 73-year-old woman presented with chronic watery diarrhea
and abdominal cramping of six weeks' duration. Biopsies of the colon
revealed findings of collagenous colitis involving the endoscopically
normal right colon, and superimposed collagenous and pseudomembranous
colitis involving the rectosigmoid colon.

Endoscopically, the left colon revealed discrete ulcerative plaques,
and Clostridium difficile toxin A assay was positive. The patient partially
responded to a three-week regimen of metronidazole, and symptoms resolved
completely with subsequent steroid therapy. At follow-up endoscopy four
months later, colon biopsies demonstrated persistence of subepithelial
collagen but no pseudomembranes. The patient remained asymptomatic during
this interval. Collagenous colitis has been reported in association
with other inflammatory bowel diseases, including lymphocytic colitis,
sprue and idiopathic inflammatory bowel disease.

This unique association of collagenous colitis with an endotoxigenic
inflammatory bowel disease is presented with a review of related disease
features.

HELICOBACTER ANTIBIOTIC THERAPY

Pseudomembranous colitis after eradication of Helicobacter pylori infection
with a triple therapy.

BACKGROUND: H.pylori (H.p.) infection of the gastric mucosa is causally
related to chronic gastritis, peptic ulcer disease, MALT-lymphoma and
gastric cancer. There is also an evidence for a link between the H.p.-infection
and non-ulcer dyspepsia and even extragastric diseases. The number of
patients treated against H.p. infection is expanding. Although in the
last years the PPI-based triple therapies have been considered to be
effective and safe, in some patients, however, severe side-effects may
occur.

CASE REPORT: We report on a 86 year old female patient, who received
a one-week triple eradication therapy (metronidazole 3x400 mg/die, clarithromycin
2x250 mg/die and omeprazole 2x20 mg/die) because of non-ulcer dyspepsia.
A few days after the eradication treatment, she developed profuse watery
and bloody diarrhea and abdominal pain with distention. In stool specimens
Clostridium difficile toxin was detected. A colonoscopy showed typical
features of antibiotic associated pseudomembranous colitis. Until now,
only few reports concerning this complication have been published and
the frequency of the complication in patients eradicated for H.p. is
unknown.

The potential risk factors to develop this condition have not been
clarified. Since the complication may be potentially lethal, this severe
side-effect of the usually well-tolerated triple-therapy has to be considered
in patients suffering from profuse diarrhea and abdominal pain after
eradication therapy.

ULCERATIVE COLITIS

Ulcerative colitis complicating pseudomembranous colitis of the right
colon.

Second Department of Internal Medicine, Nagasaki University School
of Medicine, Japan.

J Gastroenterol 2002;37(4):309-12 Abstract quote

A 65-year-old man in the remission stage of ulcerative colitis developed
severe bloody diarrhea and high fever. He was treated with imipenem/cilastatin
and clindamycin for infectious enterocolitis at a local hospital, but
there was no improvement in his condition. Steroid pulse therapy was
also ineffective.

Colonoscopy revealed pseudomembranous colitis extending from the ascending
colon to the cecum, and Clostridium difficile toxin was positive in
the feces. The administration of vancomycin in addition to oral steroids
resulted in rapid improvement of the condition.

Total colonoscopy is recommended for precise diagnosis when patients
with ulcerative colitis develop intractable diarrhea during or after
antibiotic therapy.

VRE

Clostridium difficile and vancomycin-resistant enterococcus: the new
nosocomial alliance.

OBJECTIVES: The aims of this study were to determine the frequency
of the association between Clostridium difficile (C. difficile) and
vancomycin-resistant Enterococcus (VRE) and delineate the role of C.
difficile coinfection as a predictor of VRE infection versus colonization
and adverse outcome.

METHODS: Patients with both C. difficile colitis and VRE (CD/VRE) were
compared to patients with VRE alone with regard to demographics, comorbidity,
prior antibiotic therapy, and coinfection with methicillin-resistant
Staphylococcus aureus and funguria. C. difficile as a predictor of VRE
infection (VRE-I) versus colonization (VRE-C) and adverse outcome was
also studied.

RESULTS: Eighty-nine patients with VRE infection or colonization were
studied. This included 31 cases of VRE-I and 58 VRE-C. C. difficile
was isolated in 17 (19.1%) of patients; of these C. difficile was isolated
before VRE in 9 patients and after VRE in 8. The two groups did not
differ in age, residence, or comorbidity. C. difficile coinfection was
not predictive of VRE-I versus VRE-C, nor was it associated with increased
length of stay or mortality. However, the mortality rates in both groups
was high, around 30%. A significant association was noted between the
use of vancomycin and metronidazole (before the isolation of VRE) and
C. difficile coinfection (p = 0.03 and p = 0.001, respectively). A high
incidence of nosocomial coinfection with methicillin-resistant Staphylococcus
aureus, funguria, and gram-negative sepsis was noted in both groups;
the association with funguria was statistically significant (p = 0.029).

CONCLUSIONS: In conclusion, C. difficile coinfection is common in patients
with VRE infection or colonization and is significantly associated with
other nosocomial dilemmas like funguria. This may result in the emergence
of highly virulent pathogens including vancomycin-resistant C. difficile,
posing new challenges in the management of nosocomial diarrheas.

Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae
from stools submitted for Clostridium difficile testing compared to
results from a focused surveillance program.

It has been suggested that a method of performing surveillance for
vancomycin-resistant enterococci (VRE) is to screen specimens submitted
for Clostridium difficile testing. We compared this approach to our
focused surveillance program of high-risk units during October 1997
to compare the yield of VRE and multidrug-resistant Enterobacteriaceae
(MDRE) with both methods.

Of the stools submitted for C. difficile testing, 14% were positive
for VRE or MDRE, whereas rectal swabs from routine surveillance yielded
11% VRE- or MDRE-positive results. Although stools submitted for C.
difficile testing resulted in a higher percentage of positive cultures,
14 VRE- and 2 MDRE-positive patients from our high-risk population were
missed because many patients had no stool submitted for C. difficile
testing.

Therefore, while screening stools submitted for C. difficile testing
cannot replace our focused surveillance program, it appears advantageous
to assess these stools at various intervals to detect new patient reservoirs
of drug-resistant organisms that may benefit from routine surveillance
cultures.

Department of Molecular Biology and Microbiology, Tufts University
School of Medicine, Boston, MA 02211, USA.

Proc Natl Acad Sci U S A 2001 May 8;98(10):5844-9 Abstract
quote

Clostridium difficile, a causative agent of antibiotic-associated diarrhea
and its potentially lethal form, pseudomembranous colitis, produces
two large protein toxins that are responsible for the cellular damage
associated with the disease.

The level of toxin production appears to be critical for determining
the severity of the disease, but the mechanism by which toxin synthesis
is regulated is unknown. The product of a gene, txeR, that lies just
upstream of the tox gene cluster was shown to be needed for tox gene
expression in vivo and to activate promoter-specific transcription of
the tox genes in vitro in conjunction with RNA polymerases from C. difficile,
Bacillus subtilis, or Escherichia coli. TxeR was shown to function as
an alternative sigma factor for RNA polymerase.

Because homologs of TxeR regulate synthesis of toxins and a bacteriocin
in other Clostridium species, TxeR appears to be a prototype for a novel
mode of regulation of toxin genes.

OBJECTIVE: The purpose of this study was to further characterize the
CT findings of Clostridium difficile colitis and to provide for the
first time a diagnostic sensitivity, specificity, positive predictive
value, and negative predictive value to help clinicians decide whether
antibiotic treatment is warranted on the basis of CT findings while
awaiting stool test results (which may take as long as 48 hr).

MATERIALS AND METHODS: A retrospective review covering a 4-year period
was performed of the charts and CT scans of 54 symptomatic patients
with stool test results positive for C. difficile and of a control group
of 56 patients with antibiotic-associated diarrhea with stool test results
negative for C. difficile.

RESULTS: At our institution, C. difficile colitis was explicitly diagnosed
at CT in these patients with a sensitivity of 52%, specificity of 93%,
positive predictive value of 88%, and negative predictive value of 67%.
The sensitivity can be raised to 70% with no change in specificity with
more rigid adherence to diagnostic criteria of colon wall thickening
of greater than 4 mm combined with any one or more findings of pericolonic
stranding, colon wall nodularity, the "accordion" sign, or
otherwise unexplained ascites.

CONCLUSION: Although routine CT screening of antibiotic-associated
diarrhea is not advocated, the 88% positive predictive value of a diagnosis
of C. difficile colitis in those who are scanned may merit consideration
of treatment by clinicians on the basis of the CT results alone.

Toxic megacolon: role of CT in evaluation and detection of complications.

Imbriaco M, Balthazar EJ.

Department of Radiology and National Research Council, University
Federico, Naples, Italy.

Clin Imaging 2001 Sep-Oct;25(5):349-54 Abstract quote

The purpose of this study is to determine the role of CT in the evaluation
and in detecting complications in patients with toxic megacolon. A retrospective
analysis of CT findings of 18 consecutive patients with toxic megacolon
was performed.

Underlying etiology included 12 patients with pseudomembranous colitis
(PC), four patients with ulcerative colitis and two patients with cytomegalovirus
colitis. Eleven patients were HIV+. CT features, correlation with severity
of disease and development of complications were analyzed. Colonic dilatation
with intraluminal air and/or fluid with a distorted colonic contour
or an ahaustral pattern was seen in all patients. In four patients (22%),
CT depicted complications-two colonic perforations and two septic thrombosis
of the portal system. Six patients died (33%), three of whom had the
above complications. The presence and degree of submucosal edema (accordion
sign, target sign), wall thickening, degree of dilatation, nodular contour
and ascites did not correlate with clinical outcome.

Two thirds of patients with toxic megacolon had PC as the underlying
etiology. CT was helpful in depicting diffuse colitis, and it was instrumental
in detecting life-threatening abdominal complications, contributing
to the management of these patients. CT abnormalities cannot be used
to predict the clinical outcome unless complications develop.

LABORATORY MARKERS

LEUKOCYTOSIS

Leukocytosis as a harbinger and surrogate marker of Clostridium difficile
infection in hospitalized patients with diarrhea.

OBJECTIVES: Clostridium difficile is the etiological agent of antibiotic-associated
diarrhea and pseudomembranous colitis and is a leading cause of nosocomial
diarrhea. The objective of the study was to examine if leukocytosis
could be a harbinger and surrogate marker of C. difficile infection
in hospitalized patients.

METHODS: We retrospectively examined the medical records of 70 hospitalized
patients who presented with diarrhea of variable severity and who underwent
stool examination for enteric pathogens, including C. difficile. We
specifically recorded the white blood cell count and the pattern and
severity of leukocytosis in two groups of patients--those who were C.
difficile-positive and those who were negative.

RESULTS: Leukocytosis was common in C. difficile-positive patients,
compared to in C. difficile-negative patients (mean 15,800/mm3 vs 7700/mm3,
p < 0.01). Review of the 35 C. difficile-positive patients revealed
three patterns: Pattern A) sudden WBC increase coinciding with the onset
of symptoms suggestive of C. difficile; Pattern B) unexplained leukocytosis
preceding the appearance of C. difficile-related diarrhea and serving
as a harbinger of the infection; and Pattern C) worsening of pre-existing
leukocytosis as a surrogate marker of C. difficile infection. Treatment
with metronidazole led to amelioration of symptoms and normalization
of the leukocyte count in all cases.

CONCLUSIONS: Infection with C. difficile should be considered in the
differential diagnosis of sudden onset of leukocytosis in hospitalized
patients previously or concurrently treated with antibiotics. Doing
so may obviate the need for expensive and time-consuming tests for other
etiologies.

PCR

Diagnosis and monitoring of Clostridium difficile infections with the
polymerase chain reaction.

Kuhl SJ, Tang YJ, Navarro L, Gumerlock PH, Silva J Jr.

Department of Internal Medicine, School of Medicine, University
of California, Davis, Sacramento

Clin Infect Dis 1993 Jun;16 Suppl 4:S234-8 Abstract quote

Toxigenic Clostridium difficile is the etiologic agent of pseudomembranous
colitis.

We have developed an assay system for the rapid direct detection of
toxigenic C. difficile in human stool samples. After DNA extraction,
polymerase chain reaction (PCR) amplification is undertaken with primers
targeting specific sequences in the C. difficile 16S rRNA gene.

Next, toxigenic strains of C. difficile are distinguished from nontoxigenic
strains by PCR amplification of toxin A and/or B gene sequences. This
study included 12 patients with C. difficile colitis, seven of whom
had clinical relapses after discontinuation of vancomycin therapy.

We detected toxigenic C. difficile in stools from four (57%) of these
seven patients before relapse--at a time when no toxin B was detectable
in stools and results of anaerobic culture were negative. The PCR assay
is 100-fold more sensitive than anaerobic culture methods. The course
of the infection in one patient (both during and after therapy) was
monitored by the PCR technique. The multigene analysis approach permitted
the detection of colonization with a nontoxigenic strain when this patient's
relapses had cleared. This clinically applicable assay allows earlier
detection of infection with toxigenic C. difficile. The result is more
timely therapeutic intervention.

Laboratory diagnosis of toxigenic Clostridium difficile by polymerase
chain reaction: presence of toxin genes and their stable expression
in toxigenic isolates from Japanese individuals.

Department of Bacteriology, School of Medicine, Kanazawa University,
Japan.

J Gastroenterol 1999 Feb;34(1):41-5 Abstract
quote

Clostridium difficile causes pseudomembranous colitis and antibiotic-associated
diarrhea. The definitive diagnosis of C. difficile infection is finally
accomplished by the isolation of toxigenic C. difficile. However, only
a small number of Japanese clinical laboratories are able to reach a
definitive diagnosis of C. difficile infection, probably because simple
reliable assays for toxins in the isolates are not available.

In this study, we examined the compatibility of a polymerase chain
reaction (PCR) assay and tissue culture assay to identify toxigenic
C. difficile, in toxigenic and nontoxigenic C. difficile isolates from
Japanese patients and healthy carriers. The specificity of PCR primers
was demonstrated by restriction endonuclease digestion and seminested
PCR in C. difficile VPI 10463 strain. No PCR product was amplified in
the eight other clostridial species used to check the specificity of
the PCR assay. The detection limit was 10(3) cells. Both toxin A and
toxin B genes (the genes encoding the major virulence factors of C.
difficile) were detected in 58 toxigenic C. difficile isolates, which
showed a wide range of cytotoxic activity in tissue culture assays.
Neither of the toxin genes was carried by 40 nontoxigenic strains of
C. difficile.

The results of this study strongly suggest that a definitive diagnosis
of C. difficile infection can be accomplished by PCR detection of the
toxin genes rather than by tissue culture assay of isolates.

Two molecular fingerprinting techniques, pulsed-field gel electrophoresis
(PFGE) and amplified fragment length polymorphism (AFLP), were used
to investigate the epidemiological relatedness among Clostridium difficile
isolates from suspected outbreaks in three general hospitals.

Analysis by PFGE yielded inconclusive data as a result of extensive
DNA degradation. Although this degradation could be prevented to a certain
extent by the inclusion of thiourea in the electrophoresis buffer, the
weak DNA banding patterns obtained in this way were still far from optimal.
AFLP data were obtained by using fluorescently labeled PCR primers and
analysis on an ABI PRISM automated DNA analysis platform. AFLP analysis
yielded high resolution and highly reproducible DNA fingerprinting patterns
from which the epidemiological relatedness among the isolates could
easily be determined. AFLP results could be readily obtained within
24 h, whereas 3 to 4 days were routinely required to complete the lengthy
PFGE protocol.

AFLP clearly proved to be a much more fail-safe fingerprinting method
for C. difficile isolates, especially for those isolates for which a
standard PFGE procedure yielded inconclusive results due to DNA degradation.

RESTRICTION ENZYME ANALYSIS

Relapse versus reinfection with Clostridium difficile.

O'Neill GL, Beaman MH, Riley TV.

Department of Microbiology, University of Western Australia, Nedlands.

Epidemiol Infect 1991 Dec;107(3):627-35 Abstract quote

Relapse of Clostridium difficile-associated diarrhoea occurs in 15-20%
of patients; however, whether relapse is due to an endogenous source
of the organism or reinfection from the environment remains unclear.

Restriction enzyme analysis (REA) of chromosomal DNA was used to type
multiple isolates from ten patients who had experienced apparent relapses.
More than half the relapses were due to infection with a new strain
of C. difficile. The remaining patients were infected with the same
strain, but whether this strain was acquired from the environment or
from endogenous sources could not be determined. Relapses with a different
strain of C. difficile could occur if an individual harboured more than
one strain in their gastrointestinal tract.

To investigate this possibility ten other patients were assessed for
carriage of multiple strains. Ten colonies from a primary culture plate
from each patient were typed by REA and tested for their ability to
produce cytotoxin. All isolates from the same patient were identical
by both methods, indicating that multiple carriage of strains may be
a rare event.

TOXIN A IMMUNOASSAY

How to detect Clostridium difficile variant strains in a routine laboratory.

Rupnik M.

Department of Biology, University of Ljubljana, Slovenia.

Clin Microbiol Infect 2001 Aug;7(8):417-20 Abstract quote

Toxin A-negative, toxin B-positive strains (A-/B+) are the best studied
examples of Clostridium difficile variant strains. In addition, there
are some other groups of variant C. difficile strains that produce both
toxins (A+/B+) or are non-cytotoxic (A-/B-) but differ from the reference
strain VPI 10463 in their toxin genes.

Here we describe two simple methods (amplification of the tcdA gene
and amplification of the binary toxin gene cdtA) which can be used in
rapid screening for variant C. difficile strains in collections or in
routine laboratories.

Fatal pseudomembranous colitis associated with a variant clostridium
difficile strain not detected by toxin A immunoassay.

BACKGROUND: Many clinical laboratories use toxin A immunoassays to
test for Clostridium difficile.

OBJECTIVE: To describe the clinical course of a patient infected with
a toxin variant strain of C. difficile that was not detected by toxin
A immunoassay; to genetically characterize this strain; and to estimate
the number of laboratories that use only toxin A immunoassays.

RESULTS: An elderly hospitalized man died of advanced pseudomembranous
colitis. Four stool specimens submitted over a 2-month period had tested
negative on toxin A immunoassay, but a strain of C. difficile with a
1.8-kb deletion of the toxin A gene was recovered from each specimen.
This strain, identified as restriction endonuclease analysis type CF4,
is closely related to a widely disseminated variant, toxinotype VIII.
Toxin A immunoassay was the only test being performed for detection
of C. difficile at 31 of 67 (46%) regional clinical laboratories.

CONCLUSIONS: Toxin A variant strains of C. difficile cause serious
disease and are undetectable in clinical laboratories that use only
toxin A immunoassays for C. difficile testing.

OBJECTIVES: The aims of this retrospective study were 1) to determine
the ability of single-toxin assays for Clostridium difficile to detect
infection among pediatric patients with inflammatory bowel disease (IBD)
and 2) to determine the toxin assays routinely used by pediatric tertiary
care hospitals in the United States.

METHODS: Stool specimens from patients with IBD (submitted from January,
1996, to August, 1999) were evaluated for the presence of C. difficile
toxin A and toxin B. Toxin profile (toxin A alone, toxin B alone, toxin
A and B together) was compared in positive specimens. A phone interview
was conducted with representatives from laboratories in 22 pediatric
hospitals to investigate which toxin assays were routinely used.

RESULTS: A total of 697 specimens were submitted from 284 IBD patients.
In all, 81 IBD patients (28.5%) had at least one documented infection.
Toxin A assay failed to identify 41.5% of C. difficile infections. Toxin
B assay failed to detect 34.9% of C. difficile infections. Toxin profile
changed in 55% of patients with multiple infections. Of the hospitals
surveyed, 59% did not test for both toxins.

CONCLUSIONS: Single-toxin assays for C. difficile fail to detect a
significant percentage of infections. The toxins identified during one
infection are not predictive of the toxins identified in subsequent
infections. Despite this, many pediatric hospitals do not routinely
use both toxin assays to diagnose C. difficile infection. When infection
is suspected, assays for C. difficile toxin A and toxin B should be
requested.

GROSS APPEARANCE/
CLINICAL VARIANTS

CHARACTERIZATION

GENERAL

VARIANTS

EXTRACOLONIC SITES

Extracolonic manifestations of Clostridium difficile infections. Presentation
of 2 cases and review of the literature.

Jacobs A, Barnard K, Fishel R, Gradon JD.

Johns Hopkins University School of Medicine, Baltimore MD, USA.

Medicine (Baltimore) 2001 Mar;80(2):88-101 Abstract quote

Clostridium difficile is most commonly associated with colonic infection.
It may, however, also cause disease in a variety of other organ systems.
Small bowel involvement is often associated with previous surgical procedures
on the small intestine and is associated with a significant mortality
rate (4 of 7 patients).

When associated with bacteremia, the infection is, as expected, frequently
polymicrobial in association with usual colonic flora. The mortality
rate among patients with C. difficile bacteremia is 2 of 10 reported
patients. Visceral abscess formation involves mainly the spleen, with
1 reported case of pancreatic abscess formation. Frequently these abscesses
are only recognized weeks to months after the onset of diarrhea or other
colonic symptoms. C. difficile-related reactive arthritis is frequently
polyarticular in nature and is not related to the patient's underlying
HLA-B27 status. Fever is not universally present. The most commonly
involved joints are the knee and wrist (involved in 18 of 36 cases).
Reactive arthritis begins an average of 11.3 days after the onset of
diarrhea and is a prolonged illness, taking an average of 68 days to
resolve.

Other entities, such as cellulitis, necrotizing fasciitis, osteomyelitis,
and prosthetic device infections, can also occur. Localized skin and
bone infections frequently follow traumatic injury, implying the implantation
of either environmental or the patient's own C. difficile spores with
the subsequent development of clinical infection. It is noteworthy that
except for cases involving the small intestine and reactive arthritis,
most of the cases of extracolonic C. difficile disease do not appear
to be strongly related to previous antibiotic exposure. The reason for
this is unclear.

We hope that clinicians will become more aware of these extracolonic
manifestations of infection, so that they may be recognized and treated
promptly and appropriately. Such early diagnosis may also serve to prevent
extensive and perhaps unnecessary patient evaluations, thus improving
resource utilization and shortening length of hospital stay.

FULMINANT

Fulminant Clostridium difficile: an underappreciated and increasing
cause of death and complications.

OBJECTIVE: To review the epidemiology and characteristics of patients
who died or underwent colectomy secondary to fulminant Clostridium difficile
colitis.

SUMMARY BACKGROUND DATA: In patients with C. difficile colitis, a progressive,
systemic inflammatory state may develop that is unresponsive to medical
therapy; it may progress to colectomy or death.

METHODS: The authors reviewed 2,334 hospitalized patients with C.
difficile colitis from January 1989 to December 2000. Sixty-four patients
died or underwent colectomy for pathologically proven C. difficile colitis.

RESULTS: In 2000, the incidence of C. difficile colitis in hospitalized
patients increased from a baseline of 0.68% to 1.2%, and the incidence
of patients with C. difficile colitis in whom life-threatening symptoms
developed increased from 1.6% to 3.2%. Forty-four patients required
a colectomy and 20 others died directly from C. difficile colitis. Twenty-two
percent had a prior history of C. difficile colitis. A recent surgical
procedure and immunosuppression were common predisposing conditions.
Lung transplant patients were 46 times more likely to have C. difficile
colitis and eight times more likely to have severe disease. Abdominal
computed tomography scan correctly diagnosed all patients, whereas 12.5%
of toxin assays and 10% of endoscopies were falsely negative. Patients
undergoing colectomy for C. difficile colitis had an overall death rate
of 57%. Significant predictors of death after colectomy were preoperative
vasopressor requirements and age.

CONCLUSIONS: C. difficile colitis is a significant and increasing cause
of death. Surgical treatment of C. difficile colitis has a high death
rate once the fulminant expression of the disease is present.

Division of Gastroenterology/Hepatology, San Joaquin General Hospital,
Stockton, CA95201, USA.

J Gastroenterol 2001 Sep;36(9):629-32 Abstract quote

Pseudomembranous colitis usually presents with diarrhea in a clinical
setting of recent antibiotic use. It is uncommon to see it as a cause
of obstipation and colonic pseudo-obstruction.

We report an unusual case of an elderly woman with hypertension, congestive
heart failure, chronic obstructive pulmonary disease, chronic renal
insufficiency, and diabetes mellitus, who was admitted with fever, abdominal
pain, and distension without diarrhea. She presented with decreased
stool frequency and obstipation. She did not respond to conservative
management.

Colonoscopy revealed a picture of pseudomembranous colitis, and Clostridium
difficile toxin was positive. She responded well to metronidazole therapy.

SEPSIS

Recurrent pseudomembranous colitis as a cause of recurrent severe sepsis.

Despite effective therapy with metronidazole and vancomycin relapse
rates are 15-33 %. Although colitis is seen in critically ill patients
treated with combinations of broad-spectrum antibiotics, reports describing
severe sepsis as a result of C. difficile infection are limited. We
describe the case of recurrent severe sepsis due to recurrent local
intestinal C. difficile infection as the only identifiable etiology.
The mechanism of severe sepsis may be a derangement of the gastrointestinal
barrier function. This could result in absorption of microbes or endotoxin
or activation of inflammatory cascades in the submucosa of the intestine
or liver. In general, for successful treatment of C. difficile infections
other than anticlostridial antibiotics should be discontinuated.

However, in the present case bacterial translocation from the intestine
is an attractive explanation for severe sepsis and therefore additional
antibiotics had been administered.

HISTOLOGICAL TYPES

CHARACTERIZATION

GENERAL

The summit lesion is the earliest lesion with punctate necrosis of
the surface epithelium and overlying accumulation of fibrin, polys,
mucous and necrotic epithelial cells

The lamina propria adjoining the area of necrosis has an infiltrate
of polys and eosinophils

Later lesions develop necrosis of superficial crypts with a heavier
infiltrate of polys and a plaquelike pseudomembrane of polys, fibrin
and cellular debris which is plastered against the mucosal surface

Hallmark of most lesions is their involvement of the superficial mucosa
only

Deep denuding ulcerations may rarely occur

VARIANTS

DIFFERENTIAL DIAGNOSIS

KEY DIFFERENTIATING FEATURES

INFECTIOUS COLITIS

ISCHEMIC COLITIS

Can ischemic colitis be differentiated from C difficile colitis
in biopsy specimens?

Pseudomembranous colitis is often caused by Clostridium difficile;
however, it may also be due to ischemia.

To determine if any histologic features could be used to differentiate
C difficile from ischemia, 49 biopsies of pseudomembranous colitis (25
from patients with C difficile colitis and 24 from patients with ischemic
colitis) were coded, randomized, and evaluated for the presence of numerous
variables, including the amount and distribution of mucosal necrosis,
lamina propria hyalinization, and atrophic "micro-crypts."
Hyalinization of the lamina propria was seen in 19 cases of ischemia
but not in C difficile colitis (p < 0.0001). Atrophic-appearing micro-crypts
were seen in 18 ischemic cases and 6 C difficile cases (p < 0.0006).
Lamina propria hemorrhage, full-thickness mucosal necrosis, and a diffuse
microscopic distribution of pseudomembranes were significantly more
common in ischemia than C difficile.

Endoscopic examination identified pseudomembranes significantly more
often with C difficile than ischemia, while the endoscopic appearance
of masses or polyps was seen exclusively in cases of ischemia.

The presence of a hyalinized lamina propria appeared to be a specific
and sensitive marker for ischemia in colon biopsies with pseudomembranes.
The presence of atrophic micro-crypts, lamina propria hemorrhage, full-thickness
mucosal necrosis, diffuse involvement of all the surface of all biopsies
by pseudomembranes, and the endoscopic impression of a localized process,
polyp, or mass were also markers of ischemia, while the endoscopic identification
of diffuse pseudomembranes favored the diagnosis of C difficile.

OBJECTIVE: Severe cases of Clostridium difficile-associated diseases
with sepsis seem to be rare, as are case reports about the pathogen
involved and sepsis. Our objective was to investigate the frequency
and the clinical courses of severe cases of C. difficile-associated
diseases with a fatal outcome in our hospital.

SETTING: Teaching hospital of the University of Kiel (650 beds).

DESIGN: We reviewed retrospectively all deceased patients' charts who
had prior histological or microbiological evidence of C. difficile-associated
diarrhea (CDAD) and revised the available histological specimens of
the autopsies.

PATIENTS: Over a 4-year period (November 1994-October 1998) we diagnosed
304 cases of C. difficile-associated diseases in our hospital.

RESULTS: Eighteen of our cases with C. difficile-associated diseases
had a fatal outcome. C. difficile was not likely to be the cause of
death in two of the cases. Four of the fatal infections were community-acquired
and the reason for admission to the hospital. CDAD is most prevalent
in elderly patients with multiple or severe underlying diseases and
tends to be overlooked. Sepsis was diagnosed in 15 of our 18 patients
with C. difficile-associated diseases.

CONCLUSION: Our study shows that severe cases of CDAD or cases with
C. difficile-associated sepsis are probably not rare. Routine testing
of fecal specimens for the presence of C. difficile toxins should be
considered not only in nosocomial gastrointestinal infections but also
in community-acquired gastrointestinal infections of elderly people.

Mild or severe episodes of antibiotic-associated diarrhea (AAD) are
common side effects of antibiotic therapy. The incidence of AAD differs
with the antibiotic and varies from 5 to 25%. The major form of intestinal
disorders is the pseudomembranous colitis associated with Clostridium
difficile which occurs in 10-20% of all AAD.

In most cases of AAD discontinuation or replacement of the inciting
antibiotic by another drug with lower AAD risk can be effective. For
more severe cases involving C. difficile, the treatment of diarrhea
requires an antibiotic treatment, with glycopeptides (vancomycin) or
metronidazole. Another approach to AAD treatment or prevention is based
on the use of non-pathogenic living organisms, capable of re-establishing
the equilibrium of the intestinal ecosystem.

Several organisms have been used in treatment or prophylaxis of AAD
such as selected strains of Lactobacillus acidophilus, L. bulgaricus,
Bifidobacterium longum, and Enterococcus faecium. Another biotherapeutic
agent, a non-pathogenic yeast, Saccharomyces boulardii has been used.
In animal models of C. difficile colitis initiated by clindamycin, animals
treated with S. boulardii (at end of vancomycin therapy) had a significant
decrease in C. difficile colony-forming units, and of toxin B production.
In several clinical randomised trials (versus placebo), S. boulardii
has demonstrated its effectiveness by decreasing significantly the occurrence
of C. difficile colitis and preventing the pathogenic effects of toxins
A and B of C. difficile. It has been shown to be a safe and effective
therapy in relapses of C. difficile colitis. A good response has been
seen in children with AAD, treated by S. boulardii only.

In ICUs prevention of AAD remains based on limitation of antibiotic
overuse and spread of C. difficile or other agents of AAD should be
prevented by improved hygiene measures (single rooms, private bathrooms
for patients, use of gloves and hand washing for personnel). In addition
the increasing use of biotherapeutic agents such as S. boulardii should
permit the prevention of the major side effect of antibiotics, i.e.
AAD in at risk patients.

METRONIDAZOLE

Intravenous metronidazole for the treatment of Clostridium difficile
colitis.

METHODS: Using pharmacy and microbiology laboratory records, we retrospectively
identified patients with C. difficile colitis who received intravenous
metronidazole as initial monotherapy. To be included, patients had to
fulfill the following criteria: 1) at least six doses (equivalent to
two days of therapy) of intravenous metronidazole were administered,
2) no other potential cause for colitis was found, and 3) the diagnosis
of C. difficile colitis was firmly established. For eligible patients,
five clinical parameters were assessed before and after intravenous
metronidazole.

RESULTS: Our patient group (n = 10) received an average of 13.7 (range,
6-24) doses of intravenous metronidazole as initial therapy for C. difficile
colitis. All received a dose of 500 mg three times daily. The majority
of patients with vomiting, fever, and/or abdominal pain present at the
beginning of therapy had resolution with intravenous metronidazole.
Only one patient developed a symptom (vomiting) while on therapy; however,
this eventually resolved when oral metronidazole was instituted. No
patient required colectomy for refractory colitis or developed toxic
megacolon. No patient, including those on prolonged courses, developed
toxicity related to intravenous metronidazole such as peripheral neuropathy.

Decompressive colonoscopy with intracolonic vancomycin administration
for the treatment of severe pseudomembranous colitis.

Shetler K, Nieuwenhuis R, Wren SM, Triadafilopoulos G.

Sections of Gastroenterology and Gastroenterology Division, Stanford
University School of Medicine, Stanford, California, USA.

Surg Endosc 2001 Jul;15(7):653-9 Abstract quote

BACKGROUND: We explored the potential of early decompressive colonoscopy
with intracolonic vancomycin administration as an adjunctive therapy
for severe pseudomembranous Clostridium difficile colitis with ileus
and toxic megacolon.

METHODS: We reviewed the symptoms, signs, laboratory tests, radiographic
findings, and outcomes from the medical records of seven patients who
experienced eight episodes of severe pseudomembranous colitis with ileus
and toxic megacolon. All seven patients underwent decompressive colonoscopy
with intracolonic perfusion of vancomycin.

RESULTS: Fever, abdominal pain, diarrhea, abdominal distention, and
tenderness were present in all patients. Five of seven patients were
comatose, obtunded, or confused, and six of the seven required ventilatory
support. The white blood cell count was greater than 16,000 in seven
cases (six patients). Colonoscopy showed left-side pseudomembranous
colitis in one patient, right-side colitis in one patient, and diffuse
pseudomembranous pancolitis in five patients. Two patients were discharged
with improvement. Five patients had numerous medical problems leading
to their death. Complete resolution of pseudomembranous colitis occurred
in four patients. One patient had a partial response, and two patients
failed therapy.

CONCLUSION: Colonoscopic decompression and intracolonic vancomycin
administration in the management of severe, acute, pseudomembranous
colitis associated with ileus and toxic megacolon is feasible, safe,
and effective in approximately 57% to 71% of cases.

Intracolonic use of vancomycin for treatment of clostridium difficile
colitis in a patient with a diverted colon: report of a case.